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BJD
British Journal of Dermatology
C U TA N E O U S B I O L O G Y
Intradermal injections of polyarginine-containing
immunogenic antigens preferentially elicit Tc1 and
Th1 activation and antitumour immunity
H. Mitsui, T. Okamoto, M. Kanzaki, T. Inozume, N. Shibagaki and S. Shimada
Department of Dermatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo-shi, Yamanashi 409-3898, Japan
Summary
Correspondence
Naotaka Shibagaki.
E-mail: [email protected]
Accepted for publication
9 September 2008
Key words
antigen presentation ⁄ processing, protein
transduction, skin inflammation, T cells, vaccine
Conflicts of interest
None declared.
H.M. and T.O. contributed equally to this work.
DOI 10.1111/j.1365-2133.2009.09490.x
Background We previously have shown that nona-arginine protein transduction
domain (R9-PTD) induced efficient protein-antigen (Ag) transduction of dendritic cells (DCs) in vitro, resulting in the efficient induction of strong Ag-specific
immune responses mediated by CD8+ and CD4+ T cells and in superior antitumour effects in vivo in cancer-bearing mice.
Objectives The Ag-specific immune responses caused by intradermal (i.d.) injections of R9-PTD-containing protein Ags without DC preparation were investigated. We also investigated the antitumour effects by intratumoral (i.t.)
injections of rR9-containing protein Ags.
Methods Synthesized SIINFEKL peptide, or recombinant ovalbumin fusion proteins
(rOVA, rR9-OVA), were directly injected into abdominal skin in naı̈ve C57BL ⁄6
mice. OVA-specific cytotoxic T lymphocyte (CTL) activity, serum IgG titre and
cytokine profiles were investigated. Histopathological analyses were also performed. In a cancer vaccination model, EG.7 (OVA-cDNA transfectants thymoma)
cells were inoculated intradermally in C57BL ⁄6 mice, and the antitumour effects
were evaluated by i.t. injections of rR9-OVA in a treatment setting.
Results i.d. injections of rR9-OVA into naı̈ve C57BL ⁄6 mice elicited OVA-specific
CTLs and produced IgG2-dominant immunoglobulin. The i.d. injections of rR9OVA also induced inflammatory cell infiltrates containing neutrophils, monocytes
and lymphocytes, as well as production of inflammatory cytokines such as interferon (IFN)-c, interleukin-2 and IFN-inducible protein 10, with presenting
SIINFEKL epitopes on major histocompatibility complex (MHC) class I molecules
at the injection area. i.t. injections of rR9-OVA into EG.7 tumour mass significantly suppressed tumour growth, and these effects were completely abrogated
by the depletion of CD8+ T cells. These antitumour effects were superior to
those elicited by i.t. injections of rR9-OVA-treated DCs.
Conclusions i.d. injections of rR9-containing immunogenic Ag without adjuvants
simultaneously induce dual immunological effects: the induction of Tc1- and
Th1-dominant immune responses, and the induction of inflammatory and CTLmediated immune responses at the injection area by expressing Ag epitopes on
MHC class I molecules as targets. This simple vaccination approach with R9-PTDcontaining fusion proteins might be useful as prophylactic immunotherapy for
cancer or infectious diseases.
Cytotoxic T lymphocytes (CTLs) are acquired immune effector
cells that are involved in host responses to viruses, tumours
and other intracellular pathogens. Antigen (Ag)-specific CTL
induction in vivo requires the internalization and ⁄or cross-presentation of Ag by professional Ag-presenting cells. However,
phagocytotic Ag uptake may result in only a small amount of
Ag epitopes on major histocompatibility complex (MHC) class
I molecules due to inefficient Ag cross-presentation.
Protein transduction domains (PTDs) ⁄cell-penetrating peptides (CPPs) are short stretches of cationic amino acids that
enable peptides, proteins, oligonucleotides and other reagents
efficiently to enter a variety of cell types. PTDs ⁄CPPs enter
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Journal Compilation 2009 British Association of Dermatologists • British Journal of Dermatology 2010 162, pp29–41
29
30 Polyarginine-containing immunogenic antigens and antitumour immunity, H. Mitsui et al.
cells via macropinocytosis ⁄endocytosis after binding to anionic
cell membrane components such as heparan sulphate.1–5 It has
been suggested that PTDs leak into cytoplasm after macropinocytosis, depending upon the PTD concentration.6 Thus,
PTDs ⁄CPPs offer a unique therapeutic opportunity for the
treatment of many diseases. There are some reports on the
in vivo tissue distribution of TAT-PTD ⁄cytoplasmic transduction
peptide (an arginine-rich variant of TAT-PTD) fusion proteins
through different routes of administration [portal vein, intravenous, intraperitoneal (i.p.) and oral].7,8 Previously, we
demonstrated that polyarginine (R9)-PTD, known as the most
efficacious PTD, induced more efficient protein transduction
of dendritic cells (DCs) in vitro than the other PTDs studied
including TAT-PTD; the R9-PTD-transduced DCs efficiently
induced Ag-specific immune responses mediated by CD8+
and CD4+ T cells as well as a superior antitumour response in
vivo using different model Ags.9,10 However, the clinical
potential of DC therapy is limited, because the isolation and
cultivation of DCs ex vivo require special facilities and materials,
and the treatment expense might be too great. There are some
reports that Ag-specific CTLs can be induced by using particulate carriers in vivo.11,12 If they could efficiently induce Agspecific CTLs and helper T (Th) cells, soluble protein Ags
without adjuvants would be useful in terms of safety and the
simplicity of preparation.
There are several reports describing the additional biological
effects (other than protein transduction) that are mediated by
arginine ⁄arginine-rich cationic polypeptides. These cationic
polypeptides act as antimicrobial reagents,13,14 leucocyte
chemoattractants,14,15 inducers of substance P ⁄neurokinin-116
or bronchial hyper-responsiveness,17 and indispensable nutrients for the expressions of T-cell receptors in tumour microenvironments.18 Therefore, in the present study we
intradermally injected rR9-containing ovalbumin (OVA) fusion
protein (rR9-HA-OVA) as a model Ag and then investigated
the resulting immune responses and performed histopathological analyses. Our results clearly demonstrate that intradermal
(i.d.) injections of rR9-HA-OVA into naı̈ve mice elicited quite
different Ag-specific immune responses when compared with
those of rOVA without R9-PTD. Intratumoral (i.t.) injections
of rR9-HA-OVA into EG.7-bearing mice elicited strong antitumour effects that were superior to those elicited by i.t.
injections of rR9-OVA-treated DCs. This simple vaccination
approach with R9-PTD-containing fusion proteins might be
useful as prophylactic immunotherapy for cancer or infectious
diseases.
Materials and methods
Mice and cells
Six- to 8-week-old female C57BL ⁄6 mice were purchased
from SLC Japan (Hamamatsu, Japan). Alymphoplasia immunodeficient mice (ALY ⁄NscJcl-aly ⁄aly; C57BL ⁄6 background)
were purchased from CLEA Japan (Tokyo, Japan). All animal
experiments were approved by the Institutional Review Board
of the University of Yamanashi, Faculty of Medicine. EL-4
cells (murine thymoma cells) were purchased from the
American Type Culture Collection (Manassas, VA, U.S.A.),
and OVA cDNA stable transfectants (EG.7 cells) were provided by M. Bevan (University of Washington, Seattle, WA,
U.S.A.). Cell lines were cultivated in RPMI 1640 containing
10% fetal bovine serum, glutamine and penicillin ⁄streptomycin (Invitrogen Japan, Tokyo, Japan). DCs were obtained
by culturing C57BL ⁄6 bone marrow cells in RPMI 1640 containing 5% fetal bovine serum, glutamine, penicillin ⁄streptomycin (Invitrogen), murine recombinant granulocyte ⁄
macrophage colony-stimulating factor (GM-CSF) and recombinant interleukin (IL)-4 (10 ng mL)1 each; PeproTech,
Rocky Hill, NJ, U.S.A.) for 5 days.9 After enrichment on
14Æ5% metrizamide gradients and overnight incubation, nonadherent and loosely adherent cells were harvested and used
in experiments.
Generation and characterization of recombinant protein
antigens
His6-tagged recombinant nona-arginine-haemagglutinin-OVA
(rR9-HA-OVA), rHA-OVA, rR9-HA-GFP (green fluorescent
protein) and rR9-HA-mFCRL (murine Fc receptor-like A)
constructs (Fig. S1; see Supporting Information) were generated as previously described.9,10 High expression BL21 (DE3)
(Novagen, Madison, WI, U.S.A.) transformants were selected
after blotting lysates of transformants with anti-HA monoclonal antibody (mAb) (Covance, Richmond, CA, U.S.A.).
Denatured recombinant fusion proteins were purified by
sequential Ni2+ NTA-agarose chromatography, fast protein
liquid ion exchange chromatography, and gel filtration chromatography as described.9 Proteins were stored at )70 C in
phosphate-buffered saline (PBS) ⁄10% glycerol and thawed
immediately before use. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis was performed with NuPAGE
4–12% Bis-Tris gels and MOPS running buffer (Invitrogen).
Endotoxin contamination of recombinant proteins was determined via Limulus lysate assay (BioWhittaker, Walkersville,
MD, U.S.A.). Recombinant proteins were treated with polymyxin B sulphate (50 mg mL)1; Sigma-Aldrich, St Louis,
MO, U.S.A.) before use.
Reagents
NH2-SIINFEKL-COOH (SIINFEKL) synthetic peptide and NH2RRRRRRRRR-COOH (R9) synthetic peptide were purchased
from Qiagen (Valencia, CA, U.S.A.). Native OVA protein was
purchased from Sigma-Aldrich. Complete Freund’s adjuvant
(CFA) was purchased from Invitrogen. 25.D1.16 mAb reactive
with SIINFEKL: H-2Kb complexes19 was provided by R. Germain (National Institute of Allergy and Infectious Disease,
Bethesda, MD, U.S.A.). Mouse IgG, rat IgG and hamster IgG
(isotype control), anti-Gr1 (RB6-8C5), anti-CD11b (M1 ⁄70)
and anti-CD11c (HL3) mAbs were purchased from BD Pharmingen (San Diego, CA, U.S.A.). Antimouse CD8 mAb (53-6.7)
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Polyarginine-containing immunogenic antigens and antitumour immunity, H. Mitsui et al. 31
was purchased from eBioscience (San Diego, CA, U.S.A.).
Anti-CD4 mAb (GK1.5) was provided by H. Yagita (Juntendo
University, Tokyo, Japan).
Analyses for R9-PTD-mediated GFP transduction in live
cells
For the evaluation of GFP protein transduction, DCs and EL-4
cells were incubated with rHA-GFP or rR9-HA-GFP
(100 lg mL)1) in the presence of cell culture medium in 12well plates for 1–6 h at 37 C. Then, treated cells were
washed twice with ice-cold PBS and analysed directly by flow
cytometry (FACScan; BD Pharmingen). Treated DCs or EL-4
cells were also analysed directly using a fluorescence microscope ⁄laser scanning confocal microscope (Leitz DMRBF and
Leica TCS4D; Leica, Heidelberg, Germany).
Analyses for fluorescence intensities by intradermal
injections of rGFPs in vivo
To analyse the fluorescence intensities at the injection site after
i.d. injections of rGFP proteins, naı̈ve mice were injected
intradermally on the hair-shaved right flank with rR9-HA-GFP,
rHA-GFP proteins (100 lg per mouse) or rHA-GFP (100 lg
per mouse) mixed with synthetic R9 peptide (10 lg per
mouse) (rHA-GFP + R9 peptide), and mean percentage of
green fluorescence intensities after i.d. injections of rGFP in
the presence of ultraviolet B were analysed by captured digital
images (0, 0Æ5, 1, 2, 4 and 6 h after i.d. injections of rGFPs)
with Adobe Photoshop software (Adobe Systems, Mountain
View, CA, U.S.A.).
Enzyme-linked immunosorbent assay for detection of
anti-OVA antibodies
Sera from immunized mice were collected just before they
were killed. To determine the levels of anti-OVA IgG in mouse
sera, 96-well flat-bottom microtitre plates were coated with
5 mg mL)1 of native OVA in 50 mmol L)1 Tris–HCl (pH 9Æ5)
for 2 h and blocked with 1 mg mL)1 of skimmed milk in
PBS ⁄1 mmol L)1 ethylenediamine tetraacetic acid for 2 h.
Serum samples were added to the wells for 2 h.9 Horseradish
peroxidase (HRP)-conjugated antimouse IgG antibody, antimouse IgG1, antimouse IgG2a, antimouse IgG2b (Jackson
ImmunoResearch, West Grove, PA, U.S.A.) and a tetramethylbenzidine substrate kit (Pierce, Rockford, IL, U.S.A.) were
used for detection. Absorbance at 450 nm was measured with
a Powerscan HT multidetection microplate reader.
Detection of cytokines from skin extracts
To profile cytokine expression at Ag injection sites, skin samples (10 · 10 mm) that were pretreated for 12 h with
SIINFEKL peptide (10 lg per mouse), rOVAs or rR9-HA-GFP
(100 lg per mouse) by i.d. injections were homogenized,
and extracts were collected. Then, multiple cytokine expression levels were detected with TransSignal mouse cytokine
antibody array Ver. 1.0 (Panomics, Fremont, CA, U.S.A.).
Images were collected and analysed with Adobe Photoshop
software. Percentage intensity = [(experimental spot ) negative control spot) ⁄(positive control spot ) negative control
spot)] · 100.
Histopathological and immunohistochemical analyses
Quantification of cytotoxic T lymphocytes
Mice were immunized intradermally in the flank with 5 · 105
DCs that had been treated with SIINFEKL peptide
(10 mmol L)1 : 1 lg mL)1 for 1 h) and with rOVAs
(300 nmol L)1: 18–20 lg mL)1 for 18 h) ex vivo on day 0.
Mice were also immunized intradermally in the flank with
SIINFEKL peptide (10 lg per mouse) or rOVAs (100 lg per
mouse) with or without 30 lL of CFA on day 0. On day 10,
popliteal lymph node cells or splenocytes were harvested and
restimulated in vitro with SIINFEKL peptide (1 lg mL)1) for
5 days. CTL activity was assessed on day 15 by using calcein
release assays performed as previously described.9,10,20 Nonadherent effector cells were harvested from in vitro restimulation cultures. EL-4 or SIINFEKL peptide-treated EL-4 cells were
labelled with calcein (Molecular Probes, Eugene, OR, U.S.A.),
washed, and added to 96-well round-bottom microtitre plates
with various numbers of effector cells. Plates were incubated
for 2Æ5 h, supernatants were recovered, and calcein release
was measured by using a Powerscan HT multidetection microplate reader (Dainippon Pharmaceutical Co., Tokyo, Japan).
Percentage specific lysis = [(experimental ) spontaneous) ⁄
(maximal ) spontaneous)] · 100. Maximal lysis was achieved
with 0Æ1% Triton X-100.
To perform the histopathological analyses of Ag-injected skin,
formaldehyde-fixed skin sections were stained with haematoxylin and eosin. Frozen skin sections were also stained with
anti-HA mAb (to detect HA-containing fusion proteins) or
25.D1.16 mAb (to detect SIINFEKL: H-2Kb complexes) and
with HRP-conjugated antimouse IgG.
For the quantitative analyses of infiltrating cell populations
at Ag injection sites, formaldehyde-fixed skin sections were
stained with various antibodies (anti-Gr1 mAb, anti-CD11b
mAb, anti-CD11c mAb, anti-CD4 mAb or anti-CD8 mAb),
and the mean number of positive cells was counted per cellinfiltrating field (400 ·) in three independent areas.
Tumour challenge study
In the tumour treatment study, EG.7 or parental EL-4 cells
(1 · 106) were injected intradermally into naı̈ve mice on day
0 followed by weekly injections of Ags by i.p., peritumoral or
i.t. injections on days 3 and 10 with SIINFEKL peptide (10 lg
per mouse), rOVAs or rR9-HA-GFP (100 lg per mouse).
Tumour sizes were determined biweekly in a blinded fashion.
In some experiments, EG.7-bearing mice were treated with
liquid nitrogen with cotton-tip at tumour mass (cryotherapy)
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Journal Compilation 2009 British Association of Dermatologists • British Journal of Dermatology 2010 162, pp29–41
32 Polyarginine-containing immunogenic antigens and antitumour immunity, H. Mitsui et al.
on days 3 and 10. Tumour index (in millimetres) = square
root (length · width). For CD4+ and CD8+ T-cell depletion
in vivo, mice received anti-CD4 (GK1.5) and ⁄or anti-CD8 (536.7) mAb or control rat IgG intraperitoneally twice (500 lg
1 day before the first immunization and 250 lg 1 day before
the second immunization). Administration of anti-CD4 and ⁄or
anti-CD8 mAb selectively depleted > 95% of the relevant lymphocyte subsets (assessed in the spleen 5 days after the second
dose of mAb), whereas normal rat IgG had no effect (data not
shown).
Statistical analysis
Differences between the means of the experimental groups
were analysed by Student’s t-test. P < 0Æ05 was considered
statistically significant.
Results
Production and characterization of recombinant nonaarginine-containing fusion proteins
Average green fluorescence intensities (%)
We previously demonstrated that rR9-PTD-containing fusion
proteins (rR9-HA-OVA, rR9-HA-GFP, rR9-HA-mFCRL) (Fig. S1;
see Supporting Information) could transduce bone-marrowderived DCs in vitro.9,10 These R9-PTD-mediated protein transductions were completed within 6 h (Fig. S2; see Supporting
Information). Then we investigated the dynamics of
fluorescence intensities at the injection area after i.d. injections
of rGFPs in vivo. Our results indicated that i.d. injections of
rR9-HA-GFP remained at the injection site (right flank) for a
longer time, keeping higher fluorescence intensities (Fig. 1).
These results indicate that i.d. injections of R9-containing
protein Ags to naı̈ve mice might elicit different immune
responses than those of conventional Ags.
Immunological effects after intradermal injections of
rR9-PTD-containing OVA
Therefore, we first investigated the Ag-specific CTL activities
and titres of Ag-specific IgG after immunizing naı̈ve mice with
OVA Ags. SIINFEKL-specific CTL activities were elicited even
by i.d. injections of rR9-HA-OVA (100 lg per mouse) alone
without adjuvants against SIINFEKL-pulsed EL-4 cells (Fig. 2a),
but not (nonpulsed) EL-4 cells (data not shown). In contrast,
i.d. injections of synthetic SIINFEKL peptide (10 lg per
mouse) alone or rHA-OVA without R9-PTD (100 lg per
mouse) did not elicit detectable SIINFEKL-specifc CTL activities
(Fig. 2a). Our results also demonstrate that i.d. injections of
rR9-HA-OVA could elicit OVA-specific CTL activities in popliteal (sentinel) lymph nodes, but not in splenocytes (Fig. 2b).
We also measured the OVA-specific IgG titres from the sera of
mice immunized with OVA Ags. Titres of OVA-specific IgG
were higher from mice immunized with rR9-HA-OVA alone
than from mice immunized with rHA-OVA alone, or even
with rR9-HA-OVA-treated DCs (Fig. 2c). We next determined
the subtypes of IgG that were elicited by i.d. injections of
rR9-HA-OVA. We found that IgG2 subtypes were elevated in
sera from mice with rR9-HA-OVA treatment (Fig. 2d). Our
results indicate that i.d. injections of rR9-HA-OVA might elicit
Ag-specific CTLs and IgG2-dominant IgG production.
Histopathological analyses of antigen-injected skin
Naı̈ve mice that received i.d. injections of rR9-HA-OVA developed inflammation at the injection site, especially after the
rR9-HA-GFP
0h
rHA-GFP
120
rHA-GFP + R9 peptide
6h
rR9-HA-GFP
100
80
rHA-GFP
0h
60
6h
**
40
rHA-GFP + R9 peptide
0h
20
6h
0
0
1
2
3
4
Time course (h)
5
6
Fig 1. Dynamics of fluorescence intensities at the injection site after intradermal (i.d.) injections of rGFP proteins in vivo. Naı̈ve mice were injected
intradermally on the right flank with rR9-HA-GFP, rHA-GFP proteins (100 lg per mouse), or rHA-GFP (100 lg per mouse) mixed with synthetic
R9 peptide (10 lg per mouse) (rHA-GFP + R9 peptide), and mean percentage of green fluorescence intensities after i.d. injections of rGFP under
ultraviolet B were analysed by captured digital images (right panels: images at 0 h and 6 h after i.d. injections are shown). Results are shown as
mean ± SD, and are representative of three individual experiments. **P < 0Æ01.
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Polyarginine-containing immunogenic antigens and antitumour immunity, H. Mitsui et al. 33
(a)
rHA-OVA
SIINFEKL-peptide
25
25
20
20
15
15
15
10
10
10
5
5
5
25
rR9-HA-OVA
% lysis activity (SIINFEKL-EL4)
Direct injection
Mixture w/CFA
20
Ex-vivo treat w/DC
0
0
0
1
10
1
100
10
E/T ratio
100
1
E/T ratio
(b) 35
10
100
E/T ratio
35
Popliteal LN
30
Splenocytes
% lysis activity (EG.7)
% lysis activity (EL-4)
30
25
20
15
10
5
25
20
15
10
5
0
0
1
10
100
1
E/T ratio
10
100
E/T ratio
(c)
(d)
*
*
*
*
Fig 2. Immunological effects after intradermal (i.d.) injections of OVA antigens (Ags). (a) Induction of SIINFEKL-specific cytotoxic T lymphocytes
(CTLs). Mice were immunized intradermally in the flank either with OVA Ags alone (SIINFEKL peptide: 10 lg per mouse, left panel; rHA-OVA
protein: 100 lg per mouse, centre panel; or rR9-HA-OVA protein: 100 lg per mouse, right panel) (closed squares), with OVA Ags plus complete
Freund’s adjuvant (CFA) (closed triangles), or with 5 · 105 dendritic cells (DC) that had been pretreated with OVA Ags ex vivo (SIINFEKL peptide:
1 lg mL)1 for 1 h, rOVAs: 18–20 lg mL)1 for 18 h) (closed circles) on day 0. On day 10, popliteal lymph node (LN) cells were harvested and
restimulated in vitro with SIINFEKL peptide (1 lg mL)1) for 5 days. CTL reactivities with SIINFEKL-pulsed EL-4 or EL-4 cells (data not shown)
were assessed on day 15. (b) i.d. injections of rR9-HA-OVA could elicit OVA-specific CTL activities at popliteal (sentinel) LNs but not splenocytes.
Mice were immunized intradermally in the flank with rR9-HA-OVA (100 lg per mouse) without CFA on day 0. On day 10, popliteal LN cells or
splenocytes were harvested and restimulated in vitro with mitomycin-C-pretreated (50 lg mL)1 for 45 min) EG.7 cells for 5 days. CTL reactivities
with EL-4 cells or EG.7 cells were assessed on day 15 by calcein release assay. (c) Comparison of the level of anti-OVA IgG antibodies in
individual mice. On days 0 and 7, mice were immunized intradermally in the flank with OVA Ags as described above. On day 21, an enzymelinked immunosorbent assay (ELISA) was performed with native OVA protein-coated plates. Sera from naı̈ve mice were used as a negative control,
and sera from mice immunized with native OVA with CFA were used as positive control. *P < 0Æ05. (d) Similar ELISAs were performed with
horseradish peroxidase-conjugated anti-IgG, IgG1, IgG2a and IgG2b to compare the levels of subtypes of anti-OVA antibodies. Data are
representative of three individual experiments. *P < 0Æ05. Results are shown as mean ± SD.
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Journal Compilation 2009 British Association of Dermatologists • British Journal of Dermatology 2010 162, pp29–41
34 Polyarginine-containing immunogenic antigens and antitumour immunity, H. Mitsui et al.
second immunization (upper panels of Fig. 3). We had previously never observed severe skin inflammation after injections
of rR9-HA-OVA-treated DCs. We collected skin biopsies from
the injection area 48 h after the second immunization at
weekly intervals and performed histopathological analyses
SIINFEKL
(Fig. 3a–l). We found that the area injected with rR9-HAOVA was abundantly infiltrated by Gr1+ granulocytes (neutrophils), CD11b+ monocytes, and lymphocytes (both CD4+
and CD8+ T cells; Fig 3m). In contrast, the skin immunized
by rHA-OVA was infiltrated by some monocytes, lymphocytes
rHA-OVA
rR9-HA-OVA
rR9-HA-GFP
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
10x
400x
*
(m)
PBS
15
SIINFEKL
*
rHA-OVA
rR9-HA-OVA
Cell number/field
rR9-HA-GFP
*
10
5
*
0
Gr1
CD11b
CD11c
CD4
CD8
Monoclonal antibodies
Fig 3. Histopathological analyses of the antigen-injected skin. Naı̈ve mice were immunized in the flank with SIINFEKL peptide (10 lg per
mouse), rOVAs, or rR9-HA-GFP (100 lg per mouse). After 48 h after the second immunization at a 1-week interval, skin biopsies were
performed and vertical skin sections were stained with haematoxylin and eosin (a–h) and toluidine blue (i–l). Data are representative of three
individual experiments. (m) The mean number of positive cells by immunohistochemical staining with monoclonal antibodies was counted per
cell-infiltrating field (400 ·) at three independent areas of the same skin section. Results are shown as mean ± SD. *P < 0Æ05. PBS, phosphatebuffered saline.
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Polyarginine-containing immunogenic antigens and antitumour immunity, H. Mitsui et al. 35
We also investigated the cytokine profiles of the rR9-HA-OVA
injection area. Skin biopsies from the Ag-injected site were
homogenized, and the extracts were collected and studied
with a cytokine antibody array. In contrast to the cytokine
profile by rHA-OVA, i.d. injections of rR9-HA-OVA produced
abundant Th1-type cytokines such as interferon (IFN)-c, IL-2
and IFN-inducible protein 10 (Fig. 4). i.d. injections of rR9HA-OVA also elicited production of G-CSF, M-CSF, GM-CSF,
IL-1a and IL-4, but not of IL-5, IL-6 or IL-10. Although we
used enzyme-linked immunosorbent assay to investigate IL-17
SIINFEKL
100
90
80
80
70
70
Intensity (%)
90
60
50
40
50
40
30
20
20
10
10
0
0
G-CSF
M-CSF
GM-CSF
MIG
MIP1-alpha
IFN-gamma
TNF-alpha
IP-10
RANTES
VEGF
IL-1-alpha
IL-2
IL-4
IL-5
IL-6
IL-10
IL-12
IL-13
30
rR9-HA-OVA
100
90
80
80
70
70
Intensity (%)
90
60
50
40
rR9-HA-GFP
60
50
40
30
30
20
20
10
10
0
0
G-CSF
M-CSF
GM-CSF
MIG
MIP1-alpha
IFN-gamma
TNF-alpha
IP-10
RANTES
VEGF
IL-1-alpha
IL-2
IL-4
IL-5
IL-6
IL-10
IL-12
IL-13
Intensity (%)
100
rHA-OVA
60
G-CSF
M-CSF
GM-CSF
MIG
MIP1-alpha
IFN-gamma
TNF-alpha
IP-10
RANTES
VEGF
IL-1-alpha
IL-2
IL-4
IL-5
IL-6
IL-10
IL-12
IL-13
Intensity (%)
100
Cytokine profiles of antigen-injected skin
G-CSF
M-CSF
GM-CSF
MIG
MIP1-alpha
IFN-gamma
TNF-alpha
IP-10
RANTES
VEGF
IL-1-alpha
IL-2
IL-4
IL-5
IL-6
IL-10
IL-12
IL-13
(CD4+ T cells), eosinophils, and degranulated mast cells (as
determined by toluidine blue staining). We also examined the
skin immunized by rR9-irrelevant protein (rR9-HA-GFP).
Although the types of infiltrating cells were similar to the
infiltrating cells after rR9-HA-OVA injection, the magnitude
of inflammation was less. The skin immunized by synthetic
SIINFEKL peptide (Fig. 3a) or by synthetic R9 peptide alone
(data not shown) was infiltrated by few cells. We summarized
the numbers of infiltrating cells (per 400 · field) stained by
mAbs in Figure 3m. These results demonstrate that i.d. injections of rR9-HA-OVA could induce severe inflammatory cell
infiltration at injected skin tissue.
Fig 4. Intradermal injections of rR9-HA-OVA elicit a Th1 cytokine profile at the injection area. Naı̈ve mice were immunized in the flank with
SIINFEKL peptide (10 lg per mouse), rOVAs, or rR9-HA-GFP (100 lg per mouse). Twelve hours after antigen injections, skin samples were
collected and homogenized, and then the extracts were collected. The expression of multiple cytokines was detected with the TransSignal mouse
cytokine antibody array, and images were analysed with Adobe Photoshop software. Percentage intensity = [(experimental spot ) negative control
spot) ⁄ (positive control spot ) negative control spot)] · 100. Data are shown as mean ± SD, and are representative of two individual experiments.
2009 The Authors
Journal Compilation 2009 British Association of Dermatologists • British Journal of Dermatology 2010 162, pp29–41
36 Polyarginine-containing immunogenic antigens and antitumour immunity, H. Mitsui et al.
and IL-23 cytokine production, neither of these cytokines was
detected in skin extracts (data not shown). These results and
the results in Figure 2d (IgG2-dominant OVA-specific antibody production) indicate that i.d. injections of rR9-HA-OVA
elicit strong immune responses, especially Th1-dominant
immune responses, in the area of the injected skin.
Expression of antigen epitopes on major
histocompatibility complex class I at the antigen-injected
area
Our preliminary data demonstrated that i.d. injections of
rR9-HA-GFP could stay at the injection area for a long time,
keeping high fluorescence intensities (Fig. 1). These results
indicate that rR9-HA-OVA could be transduced into injected
dermal tissue such as fibroblasts. Our present results demonstrate that high levels of IFN-c were secreted in the injection
area (Fig. 4). Previously, we also demonstrated that high levels
of the SIINFEKL epitope were presented on the MHC class I
molecules of PTD-OVA-transduced EL-4 cells in vitro upon IFN-c
treatment;20 this high level of presentation was presumably
caused by the induction of immunoproteasomes and the upregulation of MHC class I. All these data indicate that the SIINFEKL
epitope might be presented on MHC class I molecules of dermal cells by transduction and processing of rR9-HA-OVA in vivo.
To investigate this hypothesis, skin sections from areas injected
with OVA-Ags were immunostained with 25.D1.16 mAb,
which recognizes only SIINFEKL: H-2Kb complexes.19 rR9-HA-
SIINFEKL
OVA still remained at the local injection area after 48 h, as
detected by anti-HA mAb (Fig. 5c). Interestingly, SIINFEKL:
H-2Kb complexes were detected only in skin injected with
rR9-HA-OVA (Fig. 5g, k), but not in skin injected with the
synthetic SIINFEKL peptide (Fig. 5e, i). Microscopic analyses of
skin injected with rR9-HA-OVA demonstrated that 25.D1.16positive cells were dermal fibroblasts, endothelial cells and infiltrated inflammatory cells (Fig. S3; see Supporting Information).
However, we could not determine which types of inflammatory cells expressed the SIINFEKL epitope. In contrast, the staining intensities with 25.D1.16 mAb were minimal in skin
injected with rHA-OVA (Fig. 5f, j). These results indicate that
the whole area intradermally injected with rR9-HA-OVA might
be targeted by induced OVA-specific CTLs.
Treatment of EG.7-bearing animals with rR9-HA-OVA
Next, we performed an EG.7 treatment study. First, EG.7-bearing mice were immunized on days 3 and 10 by rR9-HA-OVA
in different injection areas and by different routes, and we
compared the antitumour effects. The i.t. injections with rR9HA-OVA elicited superior antitumour effects than injections in
any other area (Fig. 6a). Next, to identify the minimal dose
needed for the maximal antitumour effect, the antitumour
effects of different doses of rR9-HA-OVA injections were compared. We found that i.t. injections of 100 lg of rR9-HAOVA per mouse optimally suppressed EG.7 tumour growth
(Fig. 6b). Next, we compared the antitumour effects elicited
rHA-OVA
rR9-HA-OVA
rR9-HA-GFP
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
Fig 5. rR9-HA-OVA stayed longer at the injection area, and the SIINFEKL epitope was expressed on major histocompatibility complex class I
molecules in the same area. Naı̈ve mice were immunized in the flank with SIINFEKL peptide (10 lg per mouse), rOVAs, or rR9-HA-GFP (100 lg
per mouse). Forty-eight hours after antigen injections, skin samples were collected, and vertical skin sections were stained with anti-HA
monoclonal antibody (mAb) (a–d) (original magnification · 10) or with 25.D1.16 mAb (e–l) (original magnification · 10 and · 400). These
skin sections were not stained by isotype control IgG (data not shown). Data are representative of at least two individual experiments.
2009 The Authors
Journal Compilation 2009 British Association of Dermatologists • British Journal of Dermatology 2010 162, pp29–41
Polyarginine-containing immunogenic antigens and antitumour immunity, H. Mitsui et al. 37
(a) 25
(b) 25
Untreated
rR9-HA-GFP
Intratumoral (20)
20
SIINFEKL
Tumor index (mm)
Intratumoral
15
10
5
Intratumoral (100)
20
Intratumoral (500)
15
10
*
Tumor index (mm)
Peritumoral
Tumor index (mm)
Untreated
I.D. (opposite flank)
20
5
0
0
5
10
15
20
0
25
(d)
0
10
rR9-HA-OVA
DC-rR9-HA-GFP
15
DC-rR9-HA-OVA
10
**
*
15
20
0
25
0
5
10
15
20
Days after EG.7 challenge
Aly/Aly mice
(e) 30
Untreated
rR9-HA-OVA + anti-CD4 mAb
rR9-HA-GFP
25
rR9-HA-OVA + anti-CD8 mAb
rHA-OVA
rR9-HA-OVA
+ anti-CD4, CD8 mAb
rR9-HA-OVA
Tumor index (mm)
20
5
Days after EG.7 challenge
Untreated
rR9-HA-OVA + isotype
25
rHA-OVA
5
Days after EG.7 challenge
Tumor index (mm)
(c) 25
Untreated
I.P.
15
10
5
20
rR9-HA-OVA (I.P.)
15
10
5
0
0
0
5
10
15
20
Days after EG.7 challenge
25
0
5
10
15
20
25
Days after EG.7 challenge
Fig 6. Antitumour effects mediated by intratumoral injections of rR9-HA-OVA in EG.7-bearing mice. EG.7 cells (1 · 106) were injected
intradermally into naı̈ve mice (a–d) or Aly ⁄ Aly mice (e) on day 0 followed by two weekly injections of antigens by intraperitoneal (a),
peritumoral (a) or intratumoral (a–e) injections on days 3 and 10. (a) rR9-HA-OVA (100 lg per mouse) was injected intraperitoneally (I.P.),
intradermally (I.D.), peritumorally or intratumorally (five mice per group). (b) rR9-HA-OVA was injected intratumorally at different doses (20,
100 or 500 lg per mouse) (five mice per group). (c) Different antigens or antigen-treated dendritic cells were injected intratumorally (five mice
per group). (d) rR9-HA-OVA was injected intratumorally on days 3 and 10. Mice also received anti-CD4 (GK1.5) and ⁄ or anti-CD8 (53-6.7)
monoclonal antibody (mAb) or control rat IgG intraperitoneally on day 2 (500 lg per mouse) and day 9 (250 lg per mouse) (five mice per
group). (e) Antigens were injected intratumorally or intraperitoneally into EG.7-bearing alymphoplastic Aly ⁄ Aly mice (five mice per group).
Tumour index (in millimetres) = square root (length · width). Data are shown as mean ± SD, and are representative of at least two individual
experiments. *P < 0Æ05, **P < 0Æ01.
by i.t. injections of different OVA-Ags (Fig. 6c). Among the
Ags tested, which included rR9-HA-OVA-treated DCs (DCrR9-HA-OVA) and cryotherapy with liquid nitrogen, rR9-HAOVA mediated the greatest antitumour effects (Table 1). We
also performed an EG.7-rechallenge study with EG.7-rejected
mice. All EG.7-rejected mice completely rejected a second
challenge with EG.7 cells (1 · 106 per mouse) (15 of 15)
and survived more than 100 days after the rechallenge (data
not shown).
We also performed treatment studies with CD4+ ⁄CD8+
cell-depletion antibodies to investigate whether the antitumour
effects were mediated by T cells. We found that the effects of
i.t. injections of rR9-HA-OVA were completely dependent on
CD8+ T cells (Fig. 6d). Interestingly, the antitumour effects
mediated by rR9-HA-OVA were partially suppressed with
depletion of CD4+ cells, and the boosting antitumour effects
after the second injection were diminished. We confirmed that
i.t. injections of rR9-HA-OVA were not cytotoxic to the tissue ⁄tumour cells in alymphoplastic immunodeficient mice
(Aly ⁄Aly; C57BL6 background) (Fig. 6e). These results indicate that antitumour effects against EG.7 cells by i.t. injections
of rR9-HA-OVA were directly mediated by CD8+ CTLs, and
CD4+ T cells supported their activities.
Antitumour effects against parental EL-4-tumour-bearing
animals with rR9-HA-OVA
Our present data indicate that i.d. injections of rR9-HA-OVA
in vivo elicited dual immunological effects. One effect is the
induction of Tc1- and Th1-dominant immune responses, and
the other effect is the induction of inflammatory responses in
the injection area due to the expression of Ag epitopes on
2009 The Authors
Journal Compilation 2009 British Association of Dermatologists • British Journal of Dermatology 2010 162, pp29–41
38 Polyarginine-containing immunogenic antigens and antitumour immunity, H. Mitsui et al.
Table 1 Summary of tumour-free survival and tumour burden after
intratumoral injections of peptides, proteins or antigen-transduced
dendritic cells (DCs). EG.7-bearing mice were also treated with liquid
nitrogen at tumour mass with cotton-tip (cryotherapy; group N) on
days 3 and 10
Tumour-free
mice
Immunogen
A: Untreated
B: SIINFEKL peptide
C: R9 peptide
D: rR9-HA-GFP
E: rR9-HA-mFCRL
F: Native OVA protein
G: rHA-OVA
H: rHA-OVA + R9
peptide
I: rR9-HA-OVA
J: DC-rR9-HA-OVA
K: DC-rR9-HA-GFP
L: rHA-OVA (opposite
flank)
M: rR9-HA-OVA
(opposite flank)
N: Liquid nitrogen
(cryotherapy)
Tumour
burden (mm)
0 ⁄ 25
0 ⁄ 10
0 ⁄5
2 ⁄ 20 (10%)
0 ⁄ 10
2 ⁄ 10 (20%)
5 ⁄ 19 (26%)
1 ⁄ 5 (20%)
20Æ00
20Æ33
17Æ16
15Æ33
16Æ58
9Æ22
8Æ90
11Æ39
±
±
±
±
±
±
±
±
2Æ86
2Æ65
3Æ57
8Æ67
3Æ97
6Æ19
6Æ94
7Æ43
19 ⁄ 25 (76%)
6 ⁄ 10 (60%)
0 ⁄ 10
0 ⁄5
2Æ78
6Æ84
18Æ52
14Æ86
±
±
±
±
5Æ16
9Æ17
2Æ24
3Æ44
0 ⁄5
12Æ92 ± 2Æ05
0 ⁄5
18Æ78 ± 1Æ97
Discussion
Data from five individual treatment studies (using the same protocol as described in Fig. 6) were pooled (day 22–25 data
reported). Tumour burdens represent the mean ± SD tumour
indices (square root of the product of horizontal and vertical
dimensions). E vs. G, P = 0Æ028; E vs. I, P < 0Æ001; G vs. I,
P = 0Æ0033.
MHC class I molecules as targets. Previously, we demonstrated
that PTD-containing OVA could be transduced into EL-4 cells
(parental cells of EG.7 that do not express OVA) in vitro and
that PTD-containing OVA-treated EL-4 cells were lysed by
OVA-specific CTLs in vitro.20 We next compared the antitumour effects against EG.7 and parental EL-4 cells mediated
by i.t. injections of fusion proteins. i.t. injections of rR9-HA-
(a)
25
Untreated
(b)
rHA-OVA
25
OVA suppressed both EG.7 and EL-4 tumour growth (Fig. 7),
but EL-4 tumour masses did progress after the injections were
discontinued (Fig. 7b). The expression levels of H-2Kb on
EG.7 and EL-4 cells were almost identical, and both cell lines
grew progressively in the same way in C57BL ⁄6 mice (data
not shown). Finally, we investigated that the SIINFEKL epitope
was presented on MHC class I molecules of tumour cells by
transduction and processing of rR9-HA-OVA in vivo by i.t.
injection. Skin sections from tumour areas injected with OVAAgs were immunostained with anti-HA mAb or 25.D1.16
mAb (Fig. 8). rR9-HA-OVA still remained at the local injection area after 48 h as detected by anti-HA mAb (Fig. 8f, i),
and SIINFEKL: H-2Kb complexes were also detected on the
(EL-4) tumour cells (Fig. 8o). Thus, rR9-HA-OVA might be
transduced into EG.7 ⁄EL-4 cells in vivo by i.t. injections. However, the expression levels of OVA might be transient in EL-4
cells due to protein degradation and ⁄or cell proliferation.
Our results demonstrate that i.d. injections of R9-PTD-containing immunogenic protein Ags in vivo simultaneously
induced dual immunological effects (induction of Ag-specific
immune responses and induction of Th1-dominant inflammation at the injection area). We have not analysed in detail
the mechanism by which i.d. injections of rR9-containing
Ags induce Ag-specific Tc1- and Th1-mediated immune
responses. However, the sentinel lymph nodes might be crucial for these responses, as i.d. injections of rR9-HA-OVA
elicited little antitumour effects in alymphoplastic (Aly ⁄Aly)
mice (Fig. 6e), and the antitumour effects mediated by i.d.
injections at the opposite site were inferior (Fig. 6a). Moreover, OVA-specific CTL activities were detected in sentinel
lymph nodes but were minimal in splenocytes (Fig. 2b). Our
results demonstrate that this simple and economical vaccination approach elicited superior antitumour effects than
DC-based immunotherapy alone (Fig. 6c, Table 1). The
superior antitumour effects mediated by direct injection of
rR9-HA-OVA might be partly due to the adjuvant effects of
Untreated
rHA-OVA
rR9-HA-OVA
Tumor index (mm)
rR9-HA-OVA
rR9-HA-GFP
20
20
rR9-HA-GFP
rR9-HA-mFCRL
15
15
10
10
**
5
**
5
0
0
0
5
10
15
20
Days after EG.7 challenge
25
0
5
10
15
20
Days after EL-4 challenge
25
Fig 7. Antitumour effects mediated by
intratumoral injections of rR9-HA-OVA in
EG.7- or EL-4-bearing mice. OVA-expressing
EG.7 (a) or parental EL-4 (b) cells (1 · 106)
were injected intradermally into naı̈ve mice
(five mice per group) on day 0, followed by
two weekly intratumoral injections of antigens
(100 lg per mouse) on days 3 and 10. Data
are shown as mean ± SD, and are
representative of three individual experiments.
**P < 0Æ01.
2009 The Authors
Journal Compilation 2009 British Association of Dermatologists • British Journal of Dermatology 2010 162, pp29–41
Polyarginine-containing immunogenic antigens and antitumour immunity, H. Mitsui et al. 39
Untreated
rHA-OVA
rR9-HA-OVA
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
(m)
(n)
(o)
25.D1.16 mAb.
Anti-HA mAb
H&E
(a)
Fig 8. Histopathological analyses of the rOVA-injected skin in EL-4-bearing mice. (Parental) EL-4 cells (1 · 106) were injected intradermally into
naı̈ve mice on day 0. EL-4-bearing mice were immunized by weekly injections of rOVAs (rHA-OVA, rR9-HA-OVA) by intratumoral injections on
days 3 and 10. Forty-eight hours after the second immunization, skin biopsies were performed and vertical skin sections were stained with
haematoxylin and eosin (a–c), anti-HA monoclonal antibody (mAb) (d–i) or 25.D1.16 mAb (j–o) (original magnification · 10 and · 400). These
skin sections were not stained by isotype control IgG (data not shown). Data are representative of at least two individual experiments.
arginine-rich cationic proteins. Recently, it was demonstrated
that F4 ⁄80+ tumour-associated macrophages (TAM) of EL-4bearing mice inhibited T-cell-mediated immune responses via
induction of T-cell apoptosis through TAM-producing arginase and NO.21 Another study found that the success of
immunotherapy by CTLs against established tumours might
be dependent upon the destruction of (CD11b+) tumour
stromal cells, which are produced by the host-cancer interaction.22 Therefore, the antitumour effects mediated by i.t.
injections of rR9-containing Ags themselves, which are superior to the effects of Ag-treated DCs ex vivo (Fig. 6c), might
be due to higher killing activities against tumour stromal
cells by the transduction of Ags into those cells and by the
expression of Ag epitopes on MHC class I molecules.
2009 The Authors
Journal Compilation 2009 British Association of Dermatologists • British Journal of Dermatology 2010 162, pp29–41
40 Polyarginine-containing immunogenic antigens and antitumour immunity, H. Mitsui et al.
We recently reported that autoantigen Fc receptor-like A
(FCRLA), which is specifically expressed in melanocytes, melanoma cells and some B-cell states (Epstein–Barr virus-transformed B cells, germinal centre centroblasts and diffuse large
B-cell lymphoma),23 could be useful as a shared target Ag in
immunotherapy for B-cell malignancies.10 We are currently
investigating the enhancement of the antitumour effects
caused by direct injections of rR9-HA-mFCRL with A20 B-cell
lymphoma cells and B16 melanoma cells, as it is difficult to
break the immunological tolerance by using conventional
autoantigens.
Temporary but superior antitumour effects against parental
EL-4 cells by i.t. injections of rR9-HA-OVA (Fig. 7b), but not
by (less immunogenic) rR9-HA-GFP, may indicate that i.t.
injections of rR9-containing immunogenic foreign Ags might
be a unique and attractive immunotherapeutic approach for
cancer treatment. Similar antitumour effects against EL-4
tumour mass were also observed by i.t. injections of another
rR9-PTD-containing immunogenic foreign Ag (Leishmania Ag;
LACK24) (our unpublished observation). Previously, several
types of tumour cells stably transfected with OVA cDNA (EL-4
thymoma, A20 B-cell lymphoma, B16 melanoma) were
utilized with OVA as a model tumour-associated Ag,25–32 as
immune tolerance might be less inducible in immunogenic
foreign Ags. We are also investigating the unique immunotherapeutic approaches provided by i.t. injections of rR9-HAOVA against A20 and B16 cells; we expect to observe the
induction of tumour-specific immune responses such as
epitope spreading.
Our present results demonstrate that R9-PTD-containing
proteins stayed for a long time at the injection area when
injected intradermally (Fig. 1), and they transduced into local
tissue cells. In contrast to rR9-HA-OVA, i.d. injections of the
less immunogenic rR9-HA-GFP elicited less inflammation at
the injection area (Figs 3d and 5d). These results indicate that
in vivo application of R9-PTD-containing fusion molecules to
local skin lesions might be useful as a novel cell-transduction
approach for molecular-targeting therapies that could deliver
medicines locally without major systemic side-effects.
In summary, this simple approach may lead to unique therapies that provide important clinical benefits to patients.
Acknowledgments
This work was supported by a grant from the Ministry of Education and Science of the Japanese Government (no.
18591241).
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Supporting Information
Additional Supporting Information may be found in the online
version of this article:
Fig S1. Amino acid sequence of nona-arginine (R9)-PTD
and schematic representation of recombinant fusion proteins
and OVA antigens.
Fig S2. Flow cytometric analyses of rGFP-treated live cells
in vitro.
Fig S3. Histopathological analyses of 25.D1.16-positive cells
at rR9-HA-OVA injection area.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials supplied
by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.
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